PROJECT SUMMARY Humans have a profound, double-edged relationship with poxviruses. On one hand, the devastating effects of smallpox are unparalleled by any other pathogen in recorded history, and its eradication is a milestone in modern medicine. On the other, poxviruses are now used as highly effective gene therapy and vaccine vectors as well as oncolytics in the treatment of cancer. Moreover, molluscum contagiosum is widespread and causes prolonged, untreatable lesions, while emerging poxviruses are a serious concern. Indeed, smallpox evolved from a rodent Taterapox virus and zoonotic poxvirus infections resulting in human-to-human transmission are being reported at an increasing frequency. In some cases smallpox vaccination does not provide protection, while live smallpox vaccines such as Vaccinia Virus (VacV) pose serious, life-threatening complications for many individuals. As such, whether it be infection by existing or future poxviruses, zoonotic infections or complications from therapeutic vectors, it is important to understand how these unusual pathogens replicate. Unlike most other double-stranded DNA viruses, poxviruses replicate in the cytoplasm of infected cells within viral factories (VFs). Encoding >200 genes that include their own polymerases, transcription factors and redox system, poxviruses exhibit remarkable self-sufficiency. Despite this, poxviruses remain absolutely dependent on gaining access to host ribosomes in order to synthesize viral proteins, representing an exploitable weakness. Indeed, we have shown previously that VacV activates the host cap-dependent translation machinery and that this can be targeted using small molecules to suppress virus replication without cytotoxicity. Our preliminary data identifies a series of new and unexpected modifications induced by VacV. This includes a viral protein that remodels mammalian Target of Rapamycin (mTOR), a key regulator of ribosome recruitment and host immune responses, displacing regulatory subunits to render mTOR constitutively active and beyond host control. In addition, mass spectrometry and dual-color live cell imaging revealed that VacV phosphorylates the small ribosomal subunit, RACK1 at unique sites not induced by other viruses or stimuli, and recruits RACK1 to VFs as they form. Moreover, we find that this modification is required for selective synthesis of late VacV proteins, but not proteins of other viruses, and is induced by a VacV kinase. Finally, proteomic analysis of ribosome complexes isolated from primary human cells further revealed that VacV induces highly selective modifications to other ribosomal proteins and to the subunit composition of ribosomes themselves. Our data suggests that this ?ribosome specialization? is important for poxvirus protein synthesis, and is dispensable to the host. Understanding how these modifications facilitate VacV protein synthesis will provide important insights into fundamental aspects of poxvirus biology as well as mechanisms of selective mRNA translation. In addition, identifying factors involved in selective viral versus host protein synthesis has the potential to uncover new therapeutic targets and approaches to combat poxvirus infection.